Impacts Of Antimicrobials On The Intestinal Microbiota And Dissemination Of OqxAB-carrying Resistant Plasmid In Chicken

The irrational use of antibiotics could affect the balance between intestinal microbiota,leading to the disruption of colonization resistance and increased susceptibility to enteric infections.At the same time,this would put intense selective pressure on the microbiota that facilitates the dissemination of antibiotic resistance and the emergence of multi-drug resistance bacteria by inducing the mutation of target genes or promoting horizontal transfer of resistance genes located on the plasmids.Additionally,heteroresistance can be an intermediate stage for completely changing from susceptibility to resistance upon exposure of bacteria to an antibiotic.OqxAB,the pasmid-encoded efflux pump confers low-level quinolone resistance.Also efflux pump AcrAB-TolC and OqxAB can synergetic contribute to tigecycline resistance.Currently,information concerning the impacts of antibiotic treatments on the intestinal microbiota and colinization resistance in chicks and the transfer of oqxAB-bearing epidemic plasmid is lacking.Meanwhile,whether oqxAB-bearing epidemic plasmid involved in heteroresistance to tigecycline in Salmonella is unknown.To address these,the following studies were carried out,so as to provide theoretical basis for the rational use of antibiotics,and for the control of dissemination of resistance determinants.In this study,four antibiotics commonly applied in poultry are used to examine the impact of antibiotics on the intestinal microflora,and subsequently the susceptibility to enteric Salmonella infection.Chickens were treatment with recommended doses of colistin,lincomycin-spectinomycin,enrofloxacin and amoxicillin in drinking water.Control groups were given antibiotic-free drinking water.Alterations in microbiota composition and colonization resistance were evaluated by cultivation-based method.Results showed that antibiotics have different effects on intestinal microflora composition.In the lincomycin-spectinomycin,amoxicillin and enrofloxacin treatment groups,microbiota perturbations were obviously.Administration of colistin has minimal impact on the intestinal microbiota with no significant changes in the detected bacterial groups,except that the bacterial load of Enterobacteriaceae was reduced.The colonization level of Salmonella Typhimurium 14028/pHXY0908 in treatment groups was higher than that of control group,indicating that application of antibiotics causing intestinal flora disorders,led to increased susceptibility to enteric infection.Horizontal transfer of plasmid-encoded multidrug-resistant determinants is a major health problem.In order to investigate the impact of antibiotic therapy on the spread of resistant plasmids,we studied the dissemination of oqxAB-bearing epidemic plasmid after exposure to enrofloxacin or florfenicol treatment in chickens inoculated with Salmonella Typhimurium harboring oqxAB-bearing plasmid pHXY0908.Fecal samples and cloacal swabs were collected for isolation of S.Typhimurium and other olaquindox-resistant Enterobacteriaceae which may have acquired the oqxAB-carrying plasmid pHXY0908 by horizontal transmission.Transconjugants were verified by PCR,replicon,conjugation and restriction fragment length polymorphism assay.oqxAB-positive transconjugants were typed by pulsed-field electrophoresis(PFGE)and multilocus sequence typing(MLST).The relative amounts of the oqxAB gene and dominant bacteria to the total intestinal microbiota were also analyzed by qPCR.Results revealed that commensal E.coli isolates from the gut of chickens acquired the oqxAB-bearing plasmid in both treated and non-treated groups,indicating that the oqxAB-bearing plasmid could be readily spread within the intestinal microflora.Additionally,in the treatment group,the average isolation rate and the frequency of occurrence of oqxAB-positive E.coli were significantly higher than those in the non-treated group,indicating that antibiotic treatment would facilitate the transfer of oqxAB-bearing plasmid.PFGE analysis showed that oqxAB-positive E.coli strains belonged to different patterns with one predominating.Moreover,MLST revealed that E.coli ST533 was closely associated with the spread of oqxAB gene.qPCR analysis indicated that antibiotic administration provided selective advantages for sustaining a significantly high level of oqxAB gene from the DNA extracted from the feces.There was also a fluctuation in the intestinal microbiota with antibiotic therapy.Bacterial heteroresistance has been identified in several combinations of bacteria and antibiotics,and it complicated the therapeutic strategies.In order to further characterize the resistance phenotye of oqxAB-bearing plasmid,MIC and population analyses were performed to evaluate population-wide susceptibility to tigecycline in Salmonella enterica serovar Typhimurium strain14028/ pHXY0908.The effects of efflux pumps on MIC levels were assessed using the efflux pump inhibitor Phe-Arg-?-naphthylamide,measuring intracellular tigecycline accumulation as well as mRNA levels of regulatory and efflux pump genes.DNA sequencing of regulatory regions were performed.S.Typhimurium ATCC14028 and plasmid-cured resistant isolate were served as control strains.Results showed that MICs of a parental strain with and without pHXY0908 as well as a plasmid-cured strain 14028/?p52 were 0.5,1,and 1 ?g/mL,respectively.Population analysis profiling(PAP)illustrated that only the pHXY0908-containg strain 14028/ pHXY0908 was heteroresistant to tigecycline.After seven daily subcultures in antibiotic-free medium,a fraction of colonies exhibited stable profiles with 4-to 8-fold increases in MIC.The frequencies of emergence of these isolates were higher in strain 14028/ pHXY0908 than either the parental or the 14028/?p52 strain.PA?N addition restored tigecycline susceptibility of these isolates and intracellular tigecycline accumulations were less in strains with reduced susceptibility in comparison with the parental strain 14028,indicating that efflux activity was responsible for reduced susceptibility to tigecycline.Heteroresistant isolates of the strain containing pHXY0908 also had elevated expression of acrB,ramA,and oqxB.DNA sequencing identified numerous mutations in RamR that have been shown to lead to ramA overexpression.Results suggested that oqxAB-bearing plasmid might engender a heteroresistant phenotype to S.Typhimurium against tigecycline and this phenomenon was probably related to overexpression of multidrug resistant efflux pumps AcrAB-TolC and OqxAB.In conculsion,application of antibiotics could affect the intestinal colonization,and microbiota perturbations after exposure to antibiotics led to increased susceptibility to enteric infection in chicks.Additionally,antibiotic treatment would facilitate the transfer of oqxAB-bearing plasmid.Results also indicated that oqxAB-bearing plasmid might engender a heteroresistant phenotype to S.Typhimurium against tigecycline by influencing the expression level of efflux pump.